PROJECT SUMMARY/ ABSTRACT Antibody-drug conjugates (ADCs) are powerful cancer therapeutics that utilize receptor-mediated endocytosis to deliver potent toxins specifically to cancer cells. ADCs are comprised of a monoclonal antibody, a toxic payload, and a linker that conjugates the two together. By combining the specificity of antibodies with a potent toxin, ADCs can minimize systemic toxicity while maximizing cancer cell killing. Although most ADCs are designed to require lysosomal processing to become active, many target- receptors are known to recycle back to the cell surface, reducing their efficacy. Our lack of knowledge of ADC intracellular trafficking is limiting our ability to design more efficacious ADCs. To understand how ADC lysosomal delivery is regulated, we performed a genome-wide CRISPR/Cas9-deletion screen to identify modifiers of an ADC developed to target B-cell malignancies. As expected, many known regulators of endo/lysosomal trafficking were identified as modulators of ADC toxicity. Surprisingly, Ubiquitin regulator X domain protein 4 (UBXN4), an ER-associated protein, was found to be required for ADC toxicity. Preliminary results show that UBXN4 deletion also protected against ADCs that utilize different toxins and linkers and have similar levels of cell-surface target antigen, suggesting that UBXN4 is regulating lysosomal delivery and/or processing of ADCs. This proposal aims to reveal the mechanisms underlying the ER-associates UBXN4?s contribution to ADC toxicity, which is likely by regulating ADC intracellular trafficking and/or lysosomal function. The findings will provide novel insights into the molecular basis of lysosomal delivery and yield critical knowledge for development of more effective anticancer drugs.